Biophysical Chemistry, Institute for Materials and Molecules, Radboud University, Heyendaalseweg 135, 6524AJ Nijmegen, The Netherlands.
Spinnovation Analytical, Toernooiveld 1, Mercator III, 6525 ED Nijmegen, The Netherlands.
J Magn Reson. 2014 Feb;239:34-43. doi: 10.1016/j.jmr.2013.11.019. Epub 2013 Dec 12.
Metabolite profiling by NMR of body fluids is increasingly used to successfully differentiate patients from healthy individuals. Metabolites and their concentrations are direct reporters of body biochemistry. However, in blood plasma the NMR-detected free-metabolite concentrations are also strongly affected by interactions with the abundant plasma proteins, which have as of yet not been considered much in metabolic profiling. We previously reported that many of the common NMR-detected metabolites in blood plasma bind to human serum albumin (HSA) and many are released by fatty acids present in fatted HSA. HSA is the most abundant plasma protein and main transporter of endogenous and exogenous metabolites. Here, we show by NMR how the two most common fatty acids (FAs) in blood plasma - the long-chain FA, stearate (C18:0) and medium-chain FA, myristate (C14:0) - affect metabolite-HSA interaction. Of the set of 18 common NMR-detected metabolites, many are released by stearate and/or myristate, lactate appearing the most strongly affected. Myristate, but not stearate, reduces HSA-binding of phenylalanine and pyruvate. Citrate signals were NMR invisible in the presence of HSA. Only at high myristate-HSA mole ratios 11:1, is citrate sufficiently released to be detected. Finally, we find that limited dilution of blood-plasma mimics releases HSA-bound metabolites, a finding confirmed in real blood plasma samples. Based on these findings, we provide recommendations for NMR experiments for quantitative metabolite profiling.
体液的 NMR 代谢物分析越来越多地用于成功区分患者和健康个体。代谢物及其浓度是体内生物化学的直接报告者。然而，在血浆中，NMR 检测到的游离代谢物浓度也受到与丰富的血浆蛋白相互作用的强烈影响，而这些相互作用在代谢物分析中尚未得到充分考虑。我们之前报道过，许多常见的 NMR 检测到的血液中的代谢物与人类血清白蛋白（HSA）结合，许多代谢物是由存在于肥胖 HSA 中的脂肪酸释放的。HSA 是最丰富的血浆蛋白，也是内源性和外源性代谢物的主要转运蛋白。在这里，我们通过 NMR 展示了两种最常见的血液中的脂肪酸（FA）-长链 FA 硬脂酸（C18:0）和中链 FA 肉豆蔻酸（C14:0）-如何影响代谢物-HSA 相互作用。在 18 种常见的 NMR 检测到的代谢物中，许多是由硬脂酸和/或肉豆蔻酸释放的，其中乳酸受影响最大。肉豆蔻酸，但不是硬脂酸，降低了苯丙氨酸和丙酮酸与 HSA 的结合。柠檬酸信号在存在 HSA 时在 NMR 中不可见。只有在高的肉豆蔻酸-HSA 摩尔比 11:1 时，柠檬酸才会被充分释放并被检测到。最后，我们发现有限稀释的血浆模拟释放了与 HSA 结合的代谢物，这一发现在真实的血浆样本中得到了证实。基于这些发现，我们为 NMR 实验提供了定量代谢物分析的建议。
